Biosensors Emerging Materials and Applications Part 13 docx

P450-Based Nano-Bio-Sensors for Personalized Medicine 471 CYP species Drugs Description Reduction potential (vs Ag/AgCl) Reference CYP1A2 Clozapine Ftorafur Antipsychotic for schizophrenia Anticancer -265mV -430mV (Antonini et al., 2003) * CYP2B4 Aminopyrine Benzphetamine Analgesic, anti- inflammatory and antipyretic Anorectic -400mV -250mV (Shumyantseva et al., 2004) (Shumyantseva et al., 2007) CYP2B6 Bupropion Cyclophospha mide Ifosfamide Lidocaine Antidepressant Anticancer and immunosuppressive Anticancer and immunosuppressive Anesthetic and antiarrhythmic -450mV -450mV -430mV -450mV (Liu et al., 2008) (Liu et al., 2008) * (Peng et al., 2008) CYP2C9 Diclofenac S-Warfarin Sulfaphenazole Tolbutamide Torsemide Analgesic and anti- inflammatory Anticoagulant Antibacterial Stimulator for insulin secretion (treatment of type II diabetes) Diuretic -41mV -36mV -41mV -37mV -19mV (Johnson et al., 2005) (Johnson et al., 2005) (Johnson et al., 2005) (Johnson et al., 2005) (Johnson et al., 2005) CYP2D6 Fluoxetine Sertaline Antidepressant Antidepressant -327mV -275mV (Iwuoha, Wilson, Howel, Mathebe, Montane-Jaime, Narinesingh, Guiseppi-Elie, 2000) (Iwuoha et al., 2007) CYP2E1 P-Nitrophenol Intermediate in the synthesis of paracetamol -300mV (Fantuzzi et al., 2004) CYP3A4 Cyclophospha mide Erythromycin Anticancer and immunosuppressive Antibiotic -450mV -625mV * (Hendricks et al., 2009) Biosensors – Emerging Materials and Applications 472 Ifosfamide Indinavir Midazolam Quinidine Progesterone Verapamil Anticancer and immunosuppressive Anti-HIV Anxiolytic, anaesthetic, sedative, anticonvulsant, and muscle relaxant Beta blocker Steroid hormone For the treatment of hypertension, angina pectoris, cardiac arrhythmia -435mV -750mV - - - -100mV * (Ignaszak et al., 2009) (Joseph et al., 2003) (Joseph et al., 2003) (Joseph et al., 2003) (Joseph et al., 2003) * Measurements obtained in studies performed by the authors, immobilizing CYPs isoforms onto carbon nanotubes. Table 3. List of CYPs used for the detection of drugs for common diseases and their reduction potential obtained with cyclic voltammetry technique. analysis of reduction peaks obtained in the cyclic-voltammograms. The electron transfer can be enhanced by electrodes nanostructuring, as using metallic or zirconium dioxide nanoparticles, carbon-nanotubes (Bistolas et al., 2005; Eggins, 2003), or other techniques for the enzyme immobilization onto the electrode surface, which have been already explained in the previous pharagraph. Different studies demonstrated that carbon-nanotubes (schematized in figure 20) promote the electron transfer between the CYP active site and the electrode and enhance biosensor sensitivity (Lyons & Keeley, 2008; Wang, 2005). In table 3 a list of a target drugs which have been detected with several CYP isoforms used as biological recognition element of biosensors is reported. So, the cytochromes P450 may be used to detect drug compounds commonly used in medical treatments by using nanoparticles or carbon nanotubes for improving the device sensitivity to reach the therapeutic ranges found in the patients’ serum. Since for the treatments some of the most common diseases, as in anti-cancer therapies, more than one drug are administrated contemporaneously, an array-based biosensor able to measure multiple- drug concentrations at the same time, by using different CYP isoforms, would be very useful and it would find several practical applications. The development of such as biosensor has to overcome several difficulties, first of all the fact that each cytochrome P450 isoform detects many drugs and that different isoforms can detect the same drug (Carrara et al., 2009). 5.2.1 Carbon Nanotube (CNTs) CNTs can be described as sp 2 carbon atoms arranged in graphitic sheets wrapped into cylinders and can have lengths ranging from tens of nanometers to several microns (Lyons & Keeley, 2008). CNTs can display metallic, semiconducting and superconducting P450-Based Nano-Bio-Sensors for Personalized Medicine 473 electron transport, possess a hollow core suitable for storing guest molecules and have the largest elastic modulus of any known material. CNTs can be made by chemical vapour deposition, carbon arc methods, or laser evaporation (Wang, 2005) and can be divided into single-walled carbon-nanotubes and multi-walled carbon-nanotubes (see figure 21). Single-walled carbon nanotubes (SWCNTs) provide good chemical stability, mechanical strength and a range of electrical conductivity. They are around ten times stronger and six times lighter than steel and they can behave as metals, semiconductors or insulators depending on their chirality and diameter (Lyons & Keeley, 2008). The chirality of the SWNT is related to the angle at which the graphene sheets are rolled up (Gooding, 2005). It has been also demonstrated (Gooding, 2005) that the conductivity properties of SWNTs can depend by the presence of catalytic particles, deriving from the fabrication process, the presence of defects in their chemical structure, ion-doping and side-wall functionalizations. Fig. 21. MWCNT and SWCNT (obtained with Nanotube Modeler © JCrystalSoft, 2010). Due to their high surface energies, SWCNTs are usually found in bundles or small aggregates composed of 10-100 tubes in parallel and in contact with each other. Multi- walled carbon nanotubes (MWCNTs) are composed of several layers of concentric graphitic cylinders. They are regarded entirely as metallic conductors, making them more suitable for electrochemical applications (Lyons & Keeley, 2008). Anyway, thanks to their electrochemical properties, both multi and single-walled carbon nanotubes could be excellent candidates for the nanostructuration of electrodes used in amperometric biosensor devices. Pre-treatments of CNTs before their deposition onto electrode surfaces, cause the formation of open-ended tubes with oxygenated functional groups, crucial for the electrochemical properties of CNTs. Because of the hydrophobicity due to the CNT walls, in aqueous solution or in polar solvents the tubes have a tendency to rapidly coagulate. Thus, dispersing tubes is usually performed in non-polar organic solvents such as in dimethylformamide (DMF) or chloroform, or with the aid of surfactants or polymers, such as Nafion. The difficulty in dispersing nanotubes in aqueous solution though has been used SWCNT MWCNT Biosensors – Emerging Materials and Applications 474 as an advantage in preparing nanotube modified electrodes where nanotubes dispersed in an organic solvent are dropped onto an electrode surface and the solvent allowed evaporating. It has been demonstrated that this kind of CNT deposition allows the nanotubes to be strongly adsorbed onto the electrode surface (Gooding, 2005). 5.2.1.1 Electron transfer CNTs-protein The best strategy for successful enzyme biosensor fabrication is to devise a configuration by which electrons can directly transfer between the redox center of the enzyme and the underlying electrode. This is achievable because the physical adsorption or covalent immobilization of enzymes onto the surface of immobilized carbon nanotubes allows a direct electrical communication between the electrode and the active site of redox-active enzymes. It has been reported (Wang, 2005) that a redox enzyme, such as the glucose oxidase or cytochrome P450, adsorbs preferentially to edge-plane sites on nanotubes. Such sites contain a significant amount of oxygenated functionalities such as hydroxyl groups or carboxylic moieties formed during the purification of CNT, which provide sites for covalent linking of CNT to biorecognition elements (or other materials) or for their integration onto polymer surface structures (Wang, 2005). Other oxygenated moieties, useful for the protein immobilization, can be also formed by the breaking of carbon- carbon bonds at the nanotube ends and at defect sites present on the side-walls. The nanotubes and enzyme molecules are of similar dimensions, which facilitate the adsorption of the enzyme without significant loss of its shape or catalytic function. It is thought that the nanotube directly reaches the prosthetic group such that the electron tunnelling distance is minimized. In this way, loss of biochemical activity and protein denaturation are prevented (Lyons & Keeley, 2008). 5.2.1.2 Nanostructuring electrode surfaces with carbon nanotubes There have been a number of approaches to randomly distributing the CNTs on electrodes by dispersing the nanotubes with a binder such as dihexadecyl-hydrogen phosphate or Nafion, forming the nanotube equivalent of a carbon paste which can be screen printed, forming a nanotube-teflon composite, drop coating onto an electrode without any binders, preparing a nanotubes paper as the electrode and abrasion onto the basal planes of pyrolytic graphite. The resultant electrode has randomly distributed tubes with no control over the alignment of the nanotubes. To better control the alignment of nanotubes a more versatile approach to producing aligned carbon nanotube arrays is by self-assembly, by using self- assembled monolayers (after the functionalization of the carboxylic-ends of CNTs with carbodiimide groups and thiols), or by directly growing of aligned nanotubes onto the surface. To do this plasma enhanced chemical vapor deposition using a nickel catalyst on a chromium coated silicon wafer can be used (Gooding, 2005). Advantages in using this method are the robustness of these electrodes and also the control over the density of the CNT film by controlling the distribution of the catalyst on the surface (Salimi et al., 2005). Figure 22 reports a comparison between SEM images of MWCNTs (on the bottom) and MWCNTs covered by 1 layer of CYP3A4 (on the top). The CNTs has been deposited by drop casting technique onto the electrode surface (30μL of a solution 1mg/ml of MWCNTs in chloroform). In the figure is visible the increase of apparent CNTs diameter due to the presence of a layer of CYP3A4 (on the top), that has been deposited by drop casting onto the CNT-surface. P450-Based Nano-Bio-Sensors for Personalized Medicine 475 Fig. 22. Comparison between SEM images of MWCNTs (on the bottom) and MWCNTs covered by 1 layer of CYP3A4 (on the top), both at 80,000X of magnification. 5.2.1.3 Enhancement of catalytic current with CNTs The chemical modification of electrode surfaces with carbon nanotubes has enhanced the activity of electrode surfaces with respect to the catalysis of biologically active species such as hydrogen peroxide, dopamine and NADH. Furthermore, multi-walled carbon nanotubes have exhibited good electronic communication with redox proteins where not only the redox center is close to the protein surface such as in Cytochrome c (Zhao et al., 2005) and horseradish peroxidase, but also when it is deeply embedded within the glycoprotein such as is found with glucose oxidase (Gooding, 2005). A recent study (Carrara et al., 2008) demonstrated the enhancement of the catalytic current in a P450-based enzyme sensor in the case of electrodes modified with MWCNT, with respect to the case of both the bare electrodes and the electrode modified with gold nanoparticles. In figure 23, a comparison between cyclic voltammograms of screen-printed bare electrode (1), electrode modified with Biosensors – Emerging Materials and Applications 476 Au nanoparticles and CYP11A1 (2) and with MWCNTs and CYP11A1 (3) is reported. In these voltammograms, a huge increase of the current peak is observable in the case of the P450 working electrode modified with gold nanoparticles respect to the bare electrode, but a further enhancement of the peak current is clearly visible in the case of MWCNTs-modified electrode with P450 (Carrara et al., 2008). Fig. 23. Cyclic voltammograms of screen-printed bare electrode (1), electrode modified with Au nanoparticles and CYP11A1 (2) and with MWCNTs and CYP11A1 (3), (Carrara et al., 2008). Reprinted from Biosensors and Bioelectronics, Vol. 24, Sandro Carrara, Victoria V. Shumyantseva, Alexander I. Archakov, Bruno Samorì, “Screen-printed electrodes based on carbon nanotubes and cytochrome P450scc for highly sensitive cholesterol biosensors”, Pages No. 148–150, Copyright (2008), with permission from Elsevier. This is the direct proof that the CNT improve the electron transfer between the electrodes and the heme groups of the cytochromes. Moreover, in the presence of MWCNT, the peak is shifted in the positive direction of the voltage axis, because P450 is easier reduced in the presence of CNT, i.e. it is easier to reduce the heme iron incorporated in the protein core. 6. Conclusions In this chapter the feasibility of cytochrome P450 as probe molecule for the design of an electrochemical biosensor for drug detection in biological fluids has been investigated. Cytochromes P450 have been chosen since they are known to be involved in the metabolism of over 1,000,000 different xenobiotic and endobiotic liphophilic substrates, in particular in the metabolism of ∼75% of all drugs. The majority of cytochromes involved in drug metabolism exhibits a certain genetic polymorphism, i.e. mutations in the CYP genes that can cause the enzyme activity to be abolished, reduced, altered or increased, with substantial consequences in drug metabolism, such as an exaggerated and undesirable pharmacological response. In order to individually optimize an ongoing drug therapy, it is required to measure the plasma concentrations of drugs or their metabolites after the P450-Based Nano-Bio-Sensors for Personalized Medicine 477 administration. This is needed for really understand how the patient metabolize drugs at the moment of the pharmacological cure. It is a strong need since most effective drug therapies for major diseases still provide benefit only to a fraction of patients, typically in the 20 to 50% range. At the present state-of-the-art the technology allows only to check the genetic predisposition of patients to metabolize a certain drug, without taking into account the many factors that can influence drug metabolism, such as lifestyle, drug-drug interactions and cytochrome P450 daily variation of the polymorphism. Although CYPs are capable in general of catalyse around 60 different classes of reactions, they have a number of features in common, such as the overall fold structure, the presence in their active site of the heme group, that allow the electron transfer to catalyze substrate oxidations and reductions, and the typical catalytic cycle which requires oxygen and electrons as part of the process of metabolism. CYPs ability to metabolize a broad spectrum of endogenous substances, e.g., fatty acids, steroid hormones, prostaglandins and in particular foreign compounds such as drugs, has made this enzyme family interesting as recognition element for biosensing. P450-based biosensors are of great interest due to the possibility of developing applications such as the detection of analytes and drugs, since the currently-available methods used for in vitro quantifying the levels of drugs in biological fluids are time-consuming and expensive. A cytochrome P450 biosensor may be a promising alternative that would provide quick measurements for drugs and metabolites with a cheap, simple to use, rapid and, in some instances, disposable equipment, which also supplies good selectivity, accuracy and sensitivity. The most suitable approach for the design of a CYP-based biosensor is the direct mediatorless electron supply from an electrode to the redox active group of the CYP, thus leading a direct flow of electrons to the enzyme. In the development of this mediator-less approach, the immobilization of CYP onto the electrode surface has to be deeply controlled in order to obtain a high probability for the protein to be attached to the electrode in a proper orientation that could optimize the electron transfer to the heme group. In this chapter different techniques for the immobilization of CYPs onto the electrode surface have been described as reported in literature, focusing the attention also on the use of nanostructures (e.g. carbon nanotubes), to improve the biosensor sensitivity. Finally, a list of drugs which have been detected with several CYP isoforms has been reported with data found in literature as well as data obtained by the authors. It is possible to conclude that cytochromes P450 may be used to detect drug compounds also reaching the therapeutic ranges found in the patients’ blood, thanks to improved performances due to nanostructured-electrodes. Since for the treatments of some of the most common diseases (e.g. in anti-cancer therapies), more than one drug are administrated contemporaneously, an array-based biosensor able to measure multiple-drug concentrations at the same time, by using different CYP isoforms, would find several practical applications and it could be a first step toward the development of a real chip for personalized-medicine. Electrode miniaturization is the next mandatory step in order to test the real feasibility of this cytochrome-based biosensor as a fully-implantable device for the detection of drugs and metabolites, as much as the evaluation of the biocompatibility of all chip’s components, with particular regard to nanostructures and cytochrome citotoxicity. Finally, kinetics studies of drugs should be carried out in order to better understand drug-drug interaction phenomena and the reactions between drugs and cytochrome P450, with regard to enzyme heterotropic kinetics and its effects on drug metabolism. Biosensors – Emerging Materials and Applications 478 A cytochrome P450-based biochip for drug detection should be a very powerful platform for personalization of drug therapy thanks to the key role of P450. However, as it has been shown in this chapter, different P450 isoforms may have the same drug compound as substrate and different drugs may be substrates of the same P450 protein. Proper strategies to develop the multiplexing P450-based biosensor arrays must be studied, considering problems due to multiple enzyme-substrate interactions and in the meanwhile maintaining high reliability and low cost of experimentation. 7. Acknowledgments The SNF Sinergia Project, code CRSII2_127547/1 and title “Innovative Enabling Micro- Nano-Bio-technologies for Implantable systems in molecular medicine and personalized therapy” financially supported this research. 8. References Aguey-Zinsou K.F; Bernhardt,P.V.; De Voss, J.J. & Slessor, K.E. Electrochemistry of P450cin: new insights into P450 electron transfer. Chem. Commun. Vol.(2003), pp.418-419 Antonini, M.; Ghisellini, P.; Pastorino, L.; Paternolli, C. & Nicolini, C. (2003). Preliminary electrochemical characterisation of cytochrome P4501A2-clozapine interaction. IEE ProC Nanobiotechnol. Vol.150, (June 2003), No.1 Armstrong, F.A. & Wilson, G.S. (2000). Recent developments in faradaic bioelectrochemistry. Electrochimica Acta. Vol.45, (2000), pp. 2623–2645 Atkins, W. (2005). Non Michaelis Menten Kinetics in Cytochrome P450-Catalized Reactions. Ann Rev Pharmacol Toxicol. Vol.45, (2005), pp 291-310 Bistolas, N.; Wollenberger, U.; Jung, C. & Scheller, F.W. (2005). 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ChemBioChem, Vol.4, (2003), pp. 82-89 [...]... for different applications, including bioprocess control, food quality control, agriculture, environment, military and in particular, for medical applications The main classes of bioreceptor elements that are applied in environmental 484 Biosensors – Emerging Materials and Applications analysis are whole cells of microorganisms, enzymes, antibodies and DNA Additionally, in the most of the biosensors described... transfer and electro catalysis Journal of Inorganic Biochemistry Vol.101, (2007), pp.859–865 482 Biosensors – Emerging Materials and Applications Shumyantseva, V.V.; Carrara, S.; Bavastrello, V.; Riley, D.J.; Bulko, T.V.; Skryabin, K.G.; Archakov, A.I & Nicolini, C (2005) Direct electron transfer between cytochrome P450scc and gold nanoparticles on screen-printed rhodium–graphite electrodes Biosensors and. .. (ion-selective electrode) (5), potentiostat and data recorder (6) Standard sample and discard sample are numbered in Figure 5 as 1 and 7, respectively Silicone tubing was used for connections Fig 6 Schematic set-up for biosensor system for urea analysis 488 Biosensors – Emerging Materials and Applications 2.8.1 Procedure For urea analysis, calibration standards were prepared by dilution of urea stock... Wilson, G.S (1999) Electrochemical biosensors: Recommended definitions and classification Pure Appl Chem., Vol 71, pp 23332348 498 Biosensors – Emerging Materials and Applications Verma, N & Singh, M (2002) A disposable microbial based biosensor for quality control in milk Biosens Bioelectron, Vol 18, pp.1219-1228 23 Biosensors for Cancer Biomarkers Zihni Onur Uygun1 and Mustafa Kemal Sezgintürk2 1Çanakkale... modifications and alterations in protein function These gene modifications and protein changes can be useful 500 Biosensors – Emerging Materials and Applications indicators of any cancer types Besides they could be used to idetify prognosis, progression and therapeutic response of the disease(Sriwastava, 2007) For development, evaluating, and validating biomarkers guding principles known as the five-phase... diagnosis of lung cancer in recent years, based on the methods researched and began to develop rapidly (Heighway et al., 2002, Hirsch et al., 2001 and Qiao et al., 1997; Singhalet al., 2005; Belinsky, 2004) Annexin II, also known as Annexin encoded by the gene ANXA2 used in the diagnosis of lung 504 Biosensors – Emerging Materials and Applications Table Of Electrochemical Transducers For Detection of AFP... this study, for urease biosensor development, the urease was covalent immobilized on nylon screen by glutaraldehyde and the ammonia produced as a result of enzymatic reaction was monitored by potentiometry The enzyme employed was from a rather non- 496 Biosensors – Emerging Materials and Applications Storage time (days) Linearity range of urea concentration (ppm) Linear equation R2 02 y = 1.698x + 2.3518... hydrolysis 486 Biosensors – Emerging Materials and Applications 2.5 Urease immobilization The enzyme (powdered jack bean) immobilization using glutaraldehyde was performed according Junior (1995) The final configuration of procedure, in brief, urease was covalently immobilized on nylon screen according to the following procedure: 0.2 g of powdered beans was placed under a nylon screen and 200 mL of... in countless parts of the body is extremely important In fact, a terrible cell turnover involving death and replacement of cell, consists everlastingly in many tissues in human body.The presantation, development, and outcome of the cancer are extremely different and complex from one patient to other Moreover the cellular and molecular levels of the cancer show the similar heterogenity and uncertainty... bean amount (0.1, 0.2, 0.3, 0.4 and 0.5 g); the pH of sample standard solution (6.0, 7.0 and 8.0) and reaction temperature (20, 25, 30 and 40°C) The assay consisted in adding the desired amount of powder in 5.0 mL of the standard solutions (several urea concentrations prepared from stock solution in potassium phosphate buffer with desired pH) and 100 µL of ISA (ionic strength adjustor buffer solution) . nanoparticles and CYP11A1 (2) and with MWCNTs and CYP11A1 (3), (Carrara et al., 2008). Reprinted from Biosensors and Bioelectronics, Vol. 24, Sandro Carrara, Victoria V. Shumyantseva, Alexander. (1), electrode modified with Biosensors – Emerging Materials and Applications 476 Au nanoparticles and CYP11A1 (2) and with MWCNTs and CYP11A1 (3) is reported. In these voltammograms,. Cyclophospha mide Erythromycin Anticancer and immunosuppressive Antibiotic -450mV -625mV * (Hendricks et al., 2009) Biosensors – Emerging Materials and Applications 472 Ifosfamide